Interplay of Affinity and Surface Tethering in Protein Recognition

Surface-tethered ligand–receptor complexes are key components in biological signaling and adhesion. They also find increasing utility in single-molecule assays and biotechnological applications. Here, we study the real-time binding kinetics between various surface-immobilized peptide ligands and their unrestrained receptors. A long peptide tether increases the association of ligand–receptor complexes, experimentally proving the fly casting mechanism where the disorder accelerates protein recognition. On the other hand, a short peptide tether enhances the complex dissociation. Notably, the rate constants measured for the same receptor, but under different spatial constraints, are strongly correlated to one another. Furthermore, this correlation can be used to predict how surface tethering on a ligand–receptor complex alters its binding kinetics. Our results have immediate implications in the broad areas of biomolecular recognition, intrinsically disordered proteins, and biosensor technology.


Supplementary
1.3. Biolayer interferometry (BLI). These measurements were conducted using an Octet RED384 instrument (FortéBio, Fremont, CA) at 24ºC. 7 The assay buffer included 150 mM NaCl, 20 mM Tris-HCl, 1 mM TCEP, 1 mg/mL bovine serum albumin (BSA), pH 7.5. Streptavidin-coated biosensors were incubated with 5 nM biotinylated SET1Win for 15 minutes. Then, the unbound peptides were washed out by rinsing the sensors in assay buffer. These experimental conditions were optimized to amplify the signal-to-noise ratio while preventing potential artifacts. These include the rebinding of receptors to the surface-immobilized peptide ligands during the dissociation phase. Prior crystallographic studies demonstrated that these ligand-receptor interactions follow a 1:1 binding model. 8,9 The association process was monitored by exposing the sensors to 3-fold serial dilutions of WDR5 proteins. The dissociation phase was probed by transferring the sensors into WDR5-free assay buffer. The association phases were fitted using the equation: 10 = ! − ( ! − " ) #$ !"# % (S1) Here, Y 0 and Y ¥ denote the responses at the initial time and infinity, respectively. k obs is the apparent first-order reaction rate constant of the association phase. t represents the cumulative time of the association reaction. The dissociation phases were fitted using the equation: = ! + ( " − ¥ ) #$ !$$ % (S2) Here, k off indicates the dissociation rate constant. Y 0 and Y ¥ are the responses at the initial time and infinity, respectively. Finally, the association rate constant, k on , was determined using the slope of the linear curve: 11,12 &'( = &) [ ] + &** (S3) Then, global fittings were achieved using several WDR5 (or WDR5 mutant) concentrations. These fittings provided the corresponding k on and k off values. Equilibrium dissociation constant values, K D , were indirectly determined using the k on and k off values (K D = k off /k on ). Three independent BLI measurements were conducted for all conditions in this study.

Surface plasmon resonance (SPR).
In this study, all SPR measurements were conducted using a Cytiva Biacore 8K instrument (Cytiva Life Sciences, Marlborough, MA), as previously reported. 1 WDR5 proteins were immobilized onto the active flow cell of each channel of a Cytiva Series S Sensor Chip CM5 (Cytiva Life Sciences). The sensor surface was then activated using an injection of 1:1 N-hydroxysuccinimide (NHS)/1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) (Cytiva Amine Coupling Kit, Cytiva Life Sciences). The protein sample was then injected across the active flow cell. Finally, both active and passive flow cells were chemically deactivated. Multicycle kinetic analyses were conducted at a flow cell temperature of 25°C and a sample compartment temperature of 20°C in a running buffer composed of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.05% Tween 20. Biacore TM Insight Evaluation Software v3 (Cytiva Life Sciences) was employed to analyze and S-5 fit the sensorgrams using a 1:1 binding interaction model to provide the association (ka) and dissociation (kd) rate constants. The KD were calculated indirectly using KD = kd/ka.

Examples of BLI sensorgrams and fittings for probing the real-time kinetics of SET1Win-WDR5 interactions.
Supplementary Fig. S1. BLI sensorgrams of ST-MLL2Win interacting with WDR5 and its mutants. 5 nM biotinylated ST-MLL2Win was loaded onto streptavidin-coated sensors for 15 minutes. 3-fold serial dilutions of WDR5 and its mutants were used to obtain individual binding curves. These sensorgrams were fitted to obtain ka-ST, kd-ST, and KD-ST (eqs. S1-S3). The fits are shown in black.

Determinations of the kinetic and equilibrium constants of the interactions of ST-SET1Win ligands with WDR5 receptors using BLI measurements.
Supplementary Table S3. Kinetic rate constants of association, ka-ST, of WDR5 and its mutants with ST-SET1Win ligands using BLI measurements. 5 nM biotinylated ST-SET1Win were loaded onto streptavidin-coated sensors for 15 minutes. 3-fold serial dilutions of WDR5 and its mutants, ranging from 0.1 µM to 9 µM, were used to obtain individual binding curves. The buffer solution contained 150 mM NaCl, 20 mM Tris-HCl, 1 mM TCEP, 1 mg/ml bovine serum albumin (BSA), pH 7.5. The binding curves were fitted using the Octet Data Analysis software. ka-ST values were provided in (M -1 s -1 ) × 10 -4 . For F133L, 3-fold serial dilutions ranging from 0. 3  Supplementary Table S4. Kinetic rate constants of dissociation, kd-ST, of WDR5 and its mutants with ST-MLL ligands using BLI measurements. The N terminus of ST-SET1Win ligands were tagged with biotin and their C-terminus were amidated. 5 nM biotinylated ST-SET1Win ligands were loaded onto streptavidin-coated sensors for 15 minutes. 3-fold serial dilutions of WDR5 and its mutants, ranging from 0.1 µM to 9 µM, were used to obtain individual binding curves. The buffer solution contained 150 mM NaCl, 20 mM Tris-HCl, 1 mM TCEP, 1 mg/ml bovine serum albumin (BSA), pH 7.5. The binding curves were fitted using the Fortebio Octet Data Analysis software. kd-ST values were provided in (s -1 ) × 10 3 . For F133L, 3fold serial dilutions ranging from 0.3 µM to 27 µM were used. Numbers represent mean ± s.d. determined from three independent BLI experimental determinations.

Supplementary Table S5. Equilibrium dissociation constants, KD-ST, of WDR5 and its mutants with ST-SET1Win ligands determined from BLI measurements.
The N terminus of ST-SET1Win ligands were tagged with biotin and their C-terminus were amidated. 5 nM biotinylated ST-SET1Win ligands were loaded onto streptavidin-coated sensors for 15 minutes. 3fold serial dilutions of WDR5 and its mutants, ranging from 0.1 µM to 9 µM, were used to obtain individual binding curves. The buffer solution contained 150 mM NaCl, 20 mM Tris-HCl, 1 mM TCEP, 1 mg/ml bovine serum albumin (BSA), pH 7.5. The binding curves were fitted using the Fortebio Octet Data Analysis software. For F133L, 3-fold serial dilutions ranging from 0.3 µM to 27 µM were used. KD-ST values were provided in nM. Numbers represent mean ± s.d. determined from three independent BLI experimental determinations.

Supplementary Fig. S3. Scatter plots of the association rate constants versus the dissociation rate constants using a linear-scale representation. (a) Data resulted from shorttether (ST) experiments. (b) Data resulted from long-tether (LT) experiments. (c) Data resulted from no tether (NT) experiments. For ST and LT experiments, MLL4Win-F133L interactions
were not quantitatively determined. Hence, they only have four points each for MLL4 (for WDR5, P216L, S218F and S175L). For NT experiments, SETd1AWin-F133L interactions were not quantitatively determined. Therefore, they only have four points for SETd1A (for WDR5, P216L, S218F and S175L). Data are provided as mean ± s.d. from three independent experimental determinations. interactions were not quantitatively determined. Hence, they only have four points each for MLL4 (for WDR5, P216L, S218F and S175L). For NT experiments, SETd1AWin-F133L interactions were not quantitatively determined. Therefore, they only have four points for SETd1A (for WDR5, P216L, S218F and S175L). Data are provided as mean ± s.d. from three independent experimental determinations.

Examples of SPR sensorgrams and fittings for probing the real-time kinetics of NT-SET1Win-WDR5 interactions.
Supplementary Fig. S5. SPR sensorgrams of NT-MLL2Win interacting with immobilized WDR5 proteins. WDR5 and its mutants were immobilized onto Cytiva Series S CM5 chips using EDC/NHS amine coupling chemistry in separate experiments. Titration series of no-tether MLL2Win (NT-MLL2Win) was injected as analyte and the corresponding association (120 sec.) and dissociation (360 sec.) curves are shown. Data for WDR5 is taken from Imran and coworkers (2021). 1 These sensorgrams were fitted to obtain ka-NT, kd-NT, and KD-NT (eqs. S1-S3). The fits are shown in black.

Supplementary Table S11. Kinetic rate constants of association, ka-NT, of immobilized WDR5 receptor and its mutants with NT-SET1Win ligands using SPR measurements.
WDR5 and its mutants were immobilized onto Cytiva Series S CM5 chips using EDC/NHS amine coupling chemistry. Titration series of the respective NT-SET1Win ligands were injected as analytes. In the case of the SETd1A-F133L binding interaction, the kinetic constants were outside the limits that could be measured by the instrument. ka-NT values were provided in (M -1 s -1 ) × 10 -4 . Values represent mean ± s.d. acquired from three independent SPR experimental determinations.
Peptide WDR5* P216L F133L S175L*** S218F *Data from Imran and co-workers. 1 **Interaction between wild-type F133L and SETd1A was detectable using a SPR measurement. However, no quantitative determinations were made due to limited time resolution of the approach. In this case, ka-NT was in the order of 10 5 M -1 s -1 assuming that the association process is in the range of values determined with the other NT-SET1Win ligands. ***The test mutant of WDR5.
Supplementary Table S12. Kinetic rate constants of dissociation, kd-NT, of WDR5 and its mutants with the NT-SET1Win ligands using SPR measurements. WDR5 proteins were immobilized onto Cytiva Series S CM5 chips using EDC/NHS amine coupling chemistry. Titration series of the respective SET1Win peptide ligands were injected as analytes. In the case of the SETd1A-F133L binding interaction, the kinetic constants were outside the limits that could be measured by the instrument. kd-NT values were provided in (s -1 ) × 10 3 . Values represent mean ± s.d. acquired from three independent SPR experimental determinations.
Peptide WDR5* P216L F133L S175L*** S218F *Data from Imran and co-workers. 1 **The upper-limit value for the detection of kd-NT using SPR experiments is explicitly specified by the instrument manufacturer. The Biacore 8K+ cannot measure rate constants of dissociation, kd-NT, faster than 0.5 s -1 . ***The test mutant of WDR5.

S-15
Supplementary Table S13. Equilibrium dissociation constants, KD-NT, of WDR5 and its mutants with the NT-SET1Win ligands using SPR measurements. Either WDR5 or its derivatives was immobilized onto Cytiva Series S CM5 chips using EDC/NHS amine coupling chemistry. Titration series of the respective NT-SET1Win ligands were injected as analytes. KD-NT was calculated directly from these kinetic rate constants using KD = kd/ka. In the case of the SETd1A-F133L binding interaction, the kinetic constants were outside the limits that could be measured by the instrument. Therefore, an affinity analysis (relative response vs. concentration dose-response curve) was used to calculate the KD-NT. KD-NT values were provided in nM. Values represent mean ± s.d. acquired from three independent SPR experimental determinations.

The 3D plots and contour maps of the dissociation rate constants under ST and LT conditions normalized to those recorded under NT conditions.
Supplementary